High throughput assays for the proteolytic activities of clostridial neurotoxins

ABSTRACT

In this application is described substrates for high-throughput assays of clostridial neurotoxin proteolytic activities. Two types of substrates are described for use in assays for the proteolytic activities of clostridial neurotoxins: (1) modified peptides or proteins that can serve as FRET substrates and (2) modified peptides or proteins that can serve as immobilized substrates. In both types a fluorescent molecules is present in the substrate, eliminating the requirement for the addition of a fluorigenic reagent. The assays described can be readily adapted for use in automated or robotic systems.

This application is a Continuation of U.S. patent application Ser. No.09/962,360, filed Sep. 25, 2001, now U.S. Pat No. 6,762,280, whichclaims benefit of U.S. Provisional Application No. 60/235,050, filedSep. 25, 2000.

INTRODUCTION

The clostridial neurotoxins consist of tetanus toxin and the sevenimmunologically distinct serotypes of botulinum neurotoxin, elaboratedby various strains of Clostridium tetani and Clostridium botulinum,respectively. They are among the most potent toxins known [Simpson, L.L. (1986) Ann. Rev. Pharmacol. Toxicol. 26: 427–453; Nieman, D. H.(1991) In: Sourcebook of Bacterial Protein Toxins (J. Alouf and J.Freer, Eds.) pp 303–348, Academic press, New York]. All references citedherein supra and infra are hereby incorporated in their entirety byreference thereto.

Nonetheless, these toxins have proven to be highly useful tools forresearch on the mechanisms of neurotransmitter release [Nieman, D. H.(1991) Trends Cell Biol. 4: 179–185; Schiavo, et al. (1994) Cell Biol.5: 221–229], and are being used as clinical drugs in humans to treat arapidly expanding group of muscle dysfunctions including strabismus,blepharospasm, cervical dystonia, and hemifacial spasm [Jancovic andBrin (1992) New Engl. J. Med. 324: 1186–1194; Kessler and Benecke (1997)Neurotoxicology 18(3): 761–770]. Although accidental botulinumintoxication is not considered a major public health threat, clostridialneurotoxins have long been recognized as potential biowarfare orbioterrorist agents [Arnon, S. S., et al. (2001) JAMA 285: 1059–1070].

The clostridial neurotoxins are synthesized by the bacteria assingle-chain proteins of Mr ˜150,000, which are subsequently cleaved byendogenous proteases to yield a light chain (Mr ˜50,000) and a heavychain (Mr ˜100,000), covalently linked to each other by a disulfide bond[Bandyopadhyay, et al. (1987) J. Biol. Chem. 262: 2660–2663]. The heavychains contain receptor-binding and translocation domains, required forentry of neurotoxin into target cells. The light chains are zincmetalloproteases, highly specific for certain proteins involved inneurotransmitter release [Montecucco and Schiavo (1994) Mol. Microbiol.13: 1–8]. Botulinum serotypes A and E cleave the protein SNAP-25, whiletetanus toxin and botulinum serotypes B, D, F, and G cleavesynaptobrevin (also called VAMP) [Pellizarri, R., et al. (1999) Philos.Trans. Royal Soc. London B. Biol. Sci. 354: 259–268]. Botulinum serotypeC cleaves both syntaxin and SNAP-25 [Foran, P., et al. (1996)Biochemistry 35: 2630–2636]. Only one peptide bond is cleaved by eachtoxin within its substrate, but this is sufficient to inactivate themechanism of neurotransmitter release. Toxicity is therefore aconsequence of clostridial neurotoxin protease activity.

In view of the widespread applications for clostridial neurotoxins inneurological research and in medicine, and because of the possibilitiesfor use as bioweapons, there is an urgent need for highly sensitive andreproducible assays that can be employed to detect the toxins inpotentially contaminated food or environmental samples, to accuratelyquantify the toxins in research reagents or preparations intended forhuman clinical use, and in the search for anti-toxin drugs. Becausebotulinum neurotoxins are proteases, it follows that practical assaysfor this activity could form the basis for detection, quantification,and drug-screening systems. However, the development of such assays hasbeen hampered by several factors: (1) As noted above, each botulinumneurotoxin will cleave only one peptide bond in a particular protein,raising the possibility that separate assays would be required for eachtoxin. (2) A considerable body of evidence has been published whichindicates that the substrate recognition requirements of clostridialneurotoxin proteases are unusually large, compared to other proteases,and include discontinuous segments of their respective neuronal targetproteins. Therefore, one would anticipate that only intact targetproteins or very long polypeptides derived therefrom can function assubstrates [for review, see Schiavo, G. et al. (1995) In: ClostridialNeurotoxins (C. Montecucco, Ed.) pp 257–274, Springer-Verlag, Berlin].(3) The clostridial neurotoxin proteases do not hydrolyze short peptidesspanning the cleavage sites, and the tertiary structures of the targetproteins are critical elements in substrate recognition [Rossetto O. etal. (1994) Nature 372: 415–416; Schiavo, G. et al. (1995) supra;Washbourne, P. et al. (1997) FEBS Lett. 418: 1–5]. (4) Relatively minorchanges in substrate structure, such as the replacement of only oneamino acid with a similar one, even at some considerable distance fromthe cleavage site, can result in complete loss of substrate function[Yamasaki, S. et al. (1994) J. Biol. Chem. 269: 12764–12772; Shone andRoberts (1994) Eur. J. Biochem 225: 263–270; Schmidt and Bostian (1997)J. Prot. Chem. 16: 19–26]. Consequently, introduction of non-naturalamino acids and/or bulky aromatic or fluorescent groups would beunlikely to result in a functional substrate.

Curently, the most commonly used methods for detecting botulinum toxinsin food and for estimating concentrations in preparations for clinicaluse are the mouse lethality bioassay [Siegel and Metzger (1979) Appl.Environ. Micrbiol. 38: 606–611] and the antibody neutralization test[Siegel (1988) J. Clin. Microbiol. 26: 2351–2356]. Both require the useof animals, can take up to four days to complete, and are inherentlyinaccurate. Furthermore, determination of botulinum toxin concentrationwith the mouse bioassay cannot be used to predict pharmacologicalpotency [Pearce, L. B. et al. (1997) Toxicon 35: 1373–1412].

Assays have been published which incorporate neurotoxin proteaseactivity as one aspect of the overall method [Ekong, T. et al. (1997)Microbiology 143: 3337–3347; Wictome, M. et al. (1999) Appl. Environ.Microbiol. 65: 3787–3792; Keller, J. et al. (1999) J. Appl. Toxicol. 19:S13–S17]. Nonetheless, they are essentially immunoassays, becausequantitation of results requires the production and use of specializedantibodies, capable of distinguishing between cleaved and uncleavedsubstrate, or between cleavage product and intact substrate. They havebeen developed only for botulinum serotypes A and B. They requiremultiple binding, elution, and washing steps, and are impractical fortrue high-throughput systems.

Other assays for the proteolytic activities of tetanus toxin and ofserotypes A and B botulinum toxins have been reported [Shone, C. et al.(1993) Eur. J. Biochem. 217: 965–971; Cornille, F. et al. (1994) Eur. J.Biochem. 222: 173–181; Schmidt and Bostian (1995) J. Prot. Chem. 14:703–708; Soleilhac, J.-M. et al. (1996) Anal. Biochem. 241: 120–127].Although these assays could be adapted to high-throughput formats, theyinclude high pressure liquid chromatography or solid-phase extractionsteps, which add significant time, complexity, and expense to theprocedures.

Recently, an assay for the proteolytic activity of type B botulinumneurotoxin has been published, which uses a fluoescence resonance energytransfer (FRET) substrate and does not require physical separation ofproducts from reactants [Anne, C. et al. (2001) Analyt. Biochem. 291:253–261]. The publication describes one substrate, suitable for use withbotulinum serotype B only. Because of the extreme specificity of eachclostridial neurotoxin for a particular peptide bond in a particularsubstrate, and the likelihood that structural modifications to any ofthe substrates will diminish or abolish cleavability (see discussionabove), nothing may be inferred from these results with respect to thesuitability of similar modifications to the substrates for the otherclostridial neurotoxins, or to modifications of type B substrate otherthan those described in the publication.

An assay for the proteolytic activity of type A botulinum toxin has beendescribed (U.S. Pat. No. 5,965,699) which can be conveniently used toquantitate, standardize, and compare different preparations of thistoxin. The method is readily adapted to high-throughput format, tosearch for compounds that inhibit botulinum protease activity (i.e.potential anti-botulinum drugs). However, the assay requires theaddition of a fluorigenic reagent (e.g. fluorescamine, but others areknown), which reacts with one of the proteolytic products to yield afluorescent derivative. Furthermore, in some cases, test samples mightcontain compounds that react directly with the fluorigenic reagent toyield fluorescent derivatives, interfering with the measurement ofbotulinum protease activity.

The assays described in this application are specifically designed toovercome or eliminate all of the difficulties and drawbacks describedabove. In view of the unusually high degree of substrate specificity andthe large substrate recognition requirements exhibited by allclostridial neurotoxins (v.s.), the extensive substrate modificationsrequired for the development of the new assays would appear to rendersuccess highly unlikely. Nonetheless, practical assays have beendeveloped, useful for a wide range of applications.

SUMMARY

It is one object of this invention to provide substrate peptidessuitable for use in fluorescence resonant energy transfer assays (FRET;also known as quenched-signal assays) for the protease activities ofclostridial neurotoxins.

It is another object of the present invention to provide substratepeptides suitable for use in solid phase assays for the proteaseactivities of clostridial neurotoxins.

It is another object of this invention to provide methods for thediscovery of compounds that inhibit or otherwise modulate clostridialneurotoxin protease activities. Such compounds may be useful inbotulinum toxin clinical applications, or as anti-clostridial neurotoxindrugs.

It is another object of this invention to provide methods fordetermining the concentrations of clostridial neurotoxins in samples(e.g. preparations of toxin intended for human clinical use) or fordetecting the presence of BoNTs in food or environmental samples, basedon the proteolytic activities of the toxins and utilizing the substratesdescribed herein.

It is another object of this invention to provide methods for detectingthe presence of clostridial neurotoxins (in food, environmental samples,etc.), based on the proteolytic activities of the toxins and utilizingthe substrates described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawing.

FIG. 1: Hydrolysis of peptide (1) by recombinant type A catalyticdomain.

FIGS. 2 a and 2 b Fluorescence solublized by different concentations ofbotulinum toxins, serotypes A, B, D, and F.

DETAILED DESCRIPTION

The present invention relates to peptides suitable for the determinationof clostridial neurotoxin proteolytic activities. The term “clostridialneurotoxins” refers to the seven serotypes of neurotoxin (types Athrough G, inclusive) produced by Clostridium botulinum, and to tetanustoxin, produced by Clostridium tetani.

The invention includes two types of substrates: (I), modified peptidesor proteins that can serve as FRET or quenched-signal substrates inassays for the proteolytic activities of clostridial neurotoxins, and(II), modified peptides or proteins that can serve as immobilizedsubstrates (i.e. covalently or otherwise bound to a solid phase) inassays for the proteolytic activities of clostridial neurotoxins. Inboth types, a fluorescent molecule is present in the substrate,eliminating the requirement for the addition of a fluorigenic reagent.In assays with type (I) substrates, toxin-catalyzed hydrolysis resultsin a proportional increase in fluorescence. Therefore, physicalseparation of cleavage products from intact substrate is not necessary.In type (II) assays, separation of products from intact substrate isaccomplished by simply transferring all or part of the soluble fractionto another container, followed by quantitation of the fluorescence inthe soluble fraction. Circumstances which favor the use of one type ofassay over the other are discussed below. The assays are called“high-throughput” because lengthy processing steps such ascentrifugation, solid-phase extraction, or chromatography are notneeded. Therefore, the assays can be readily adapted for use inautomated or robotic systems.

Circumstances favoring the use of one type of assay over the otherinclude:(1), Cost. In some instances, synthesis of a type (I) substrateis more expensive than a type (II) substrate for the same serotype. Thesubstrates for botulinum serotype A described in claims (2) and (9)illustrate this situation. The former, a FRET or type (I) substrate, ismore costly to produce than the latter. Therefore, if very large numbersof type A assays are anticipated, economics favors the use of assaysincorporating substrate (9), or another type (II) substrate. (2),Available instrumentation. The most efficient use of type (II)substrates is in multiwell arrays. However, a fluorometer capable ofreading such arrays is required. If a multiwell fluorometer is notavailable, use of a FRET substrate would be indicated. (3),Determinations of clostridial protease kinetic constants. Initial ratesof substrate hydrolyses, catalyzed by clostridial neurotoxin proteaseactivities, are most conveniently determined using FRET or type (I)substrates. Measurements of initial rates are required for calculationsof kinetic constants, such as Km, kcat (turnover number), and thebinding affinities of inhibitors or other effectors. (4), Interferenceof test samples with direct fluorescence measurements. Properties ofcertain test compounds, such as quenching, turbidity, or fluorescence,might preclude quantitation of assay results by direct fluorescencemeasurements. In this situation, use of a solid-phase or type (II) assayis indicated. At the conclusion of the incubation period, samples areremoved and the wells are washed to remove all test compounds andenzymes. The amount of uncleaved substrate still bound to each well isthen determined by incubation with trypsin, 50–100 micrograms per ml,followed by fluorescence measurements. In this situation, the presenceof an inhibitor is indicated by a higher fluorescence reading, comparedto the control, instead of a lower reading, as in direct assays. (5)Test samples bound to solid matrices. For example, combinatorialchemistry libraries are often attached to resin beads. In this case, useof a FRET substrate is sometimes more convenient than a solid-phasesubstrate.

Type (I) substrates:

The following are examples of FRET substrates for the proteolyticactivities of clostridial neurotoxins. Each contains a fluorescent group(fluorophore) on one side of the cleavage site, and a molecule thatquenches that fluorescence on the other side of the cleavage site. Uponneurotoxin-catalyzed hydrolysis, the fluorophore and quencher diffuseaway from each other, and the fluorescence signal increases inproportion to the extent of hydrolysis. Therefore, the occurrence andrate of hydrolysis may be determined by following the increase influorescence with a suitable fluorimeter. Addition of fluorigenicreagents, transfer or washing steps, or substrate immobilization are notrequired.

Sequences of type A botulinum protease substrates described in U.S. Pat.No. 5,965,699, all of which can be modified as described below for useas FRET substrates, are herein incorporated by reference.

Substrate (1) (SEQ ID NO:1) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 S N R T R I D X AN  Q  R  A  Z  R  M  L

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “Z” isS-(fluoresceinyl)-cysteine. This peptide is a substrate for theproteolytic activity of type A botulinum neurotoxin. Cleavage occursbetween residues 11 (Q) and 12 (R).

Substrate (2) (SEQ ID NO:2) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 S N R T R I D E AN  X  R  A  dcC R  M  L

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “dcC” isS-(7-dimethylamino-4-methyl-coumarin-3-carboxamidomethyl)-cysteine. Thispeptide is a substrate for the proteolytic activity of type A botulinumneurotoxin. Cleavage occurs between residues 11(N(epsilon)-(2,4-dinitrophenyl)-lysine) and 12 (R).

Substrate (3)(SEQ ID NO:3) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 L S E L D D R A DA  L  Q  A  X  A  S  Q  F  E  Z 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 S  A  A  K  L  K  R  K  Y  W  W  K  N  L  K

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “Z” isS-(fluoresceinyl)-cysteine. This peptide is a substrate for theproteolytic activity of type B botulinum neurotoxin. Cleavage occursbetween residues 17 (Q) and 18(F).

Substrate (4) (SEQ ID NO:4) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 L S E L D D R A DA  L  Q  A  G  A  S  X  F  E  dcC 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 S  A  A  K  L  K  R  K  Y  W  W  K  N  L  K

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “dcC” isS-(7-dimethylamino-4-methyl-coumarin-3-carboxamidomethyl)-cysteine. Thispeptide is a substrate for the proteolytic activity of type B botulinumneurotoxin. Cleavage occurs between residues 17(N(epsilon)-(2,4-dinitrophenyl)-lysine) and 18(F).

Substrate (5) (SEQ ID NO:5) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A Q V D E V V D IM  R  V  N  V  D  K  V  L  X  R 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 D  Q  K  L  Z  E  L  D  D  R  A  D  A  L  Q  A  G  A 39 S

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “Z” isS-(fluoresceinyl)-cysteine. This peptide is a substrate for theproteolytic activities of types D and F botulinum neurotoxins. Type Dcleaves between residues 23 (K) and 24 (L), while type F cleavesresidues 22 (Q) and 23 (K).

Substrate (6) (SEQ ID NO:6) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A Q V D E V V D IM  R  V  N  V  D  K  V  L  E  R 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 D  X  K  L  dcC E  L  D  D  R  A  D  A  L  Q  A  G  A 39S

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “dcC” isS-(7-dimethylamino-4-methyl-coumarin-3-carboxamidomethyl)-cysteine. Thispeptide is a substrate for the proteolytic activities of types D and Fbotulinum neurotoxins. Type D cleaves between residues 23 (K) and 24(L). Cleavage by type F occurs between residues 22(N(epsilon)-(2,4-dinitrophenyl)-lysine) and 23 (K).

Substrate (7) (SEQ ID NO:7) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A Q V D E V V D IM  R  V  N  V  D  K  V  L  E  R 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 D  X  K  L  mcp E  L  D D  R  A  D  A  L  Q  A  G  A 39 S

Where “X” is N(epsilon)-(2,4-dinitrophenyl)-lysine and “mcp” is2-amino-3-(7-methoxycoumarin-4-yl)-propionic acid. This peptide is asubstrate for the proteolytic activities of types D and F botulinumneurotoxins. Type D cleaves between residues 23 (K) and 24 (L). Cleavageby type F occurs between residues 22(N(epsilon)-(2,4-dinitrophenyl)-lysine) and 23 (K).

Substrate (8):

Any peptide or protein that can serve as a substrate for the proteolyticactivity of any clostridial neurotoxin, said protein or peptide havingbeen modified to contain a signal moiety on one side of the cleavagesite, and a moiety on the other side of the cleavage site that quenchesor diminishes the magnitude of that signal. When the substrate iscleaved by clostridial neurotoxin proteolytic activity, the two diffuseaway from each other and the signal increases in proportion to theamount of cleavage that has occurred. Examples of signal and quenchmoieties include, respectively: coumarin derivatives andN(epsilon)-(2,4-dinitrophenyl)-lysine; coumarin derivatives andnitrotyrosine; fluorescein and rhodamine; fluorescein andN(epsilon)-(2,4-dinitrophenyl)-lysine among others known in the art.

The general concept of FRET assays has been known for many proteases.However, knowledge provided by FRET assays for other proteases cannot beapplied directly to the development of FRET substrates for clostridialneurotoxin protease activities, due to the extreme substratespecificities, sensitivities to even minor structural changes insubstrates, and the very large substrate recognition requirements of thelatter enzymes. In view of these complex and stringent limitations,design of FRET substrates for clostridial neurotoxin proteaseactivities, with respect to types of signal and quench moieties andplacement within the substrate sequences, is not obvious.

Type (II) Substrates Claimed:

Peptides described in substrate (9)–(13) are examples of clostridialneurotoxin substrates, intended for immobilization through reaction ofthe sulfhydryl groups of the C-terminal cysteine residues in thepeptides with maleimide groups, the latter covalently bound to the wallsof multiwell plates.

Sequences of type A botulinum protease substrates described in U.S. Pat.No. 5,965,699, all of which can be modified as described in thisapplication for use as immobilized substrates, are herein incorporatedby reference.

Substrate (9) (SEQ ID NO:8) is the following peptide:

1   2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 flG G G S N R T R ID  E  A  N  Q  R  A  T  R  M  L 21 22 23 24 G  G  G  C

Where flG is N-fluoresceinyl-glycine. This peptide is a substrate forthe proteolytic activity of type A botulinum neurotoxin. Cleavage occursbetween residues 14 (Q) and 15 (R).

Substrate (10) (SEQ ID NO:9) is the following peptide:

1   2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 flG G G L S E L D DR  A  D  A  L  Q  A  G  A  S  Q 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 F  E  T  S  A  A  K  L  K  R  K  Y  W  W  K  N  L  K 3940 41 42 G  G  G  C

Where flG is N-fluoresceinyl-glycine. This peptide is a substrate forthe proteolytic activity of type B botulinum neurotoxin. Cleavage occursbetween residues 20 (Q) and 21 (F).

Substrate (11) (SEQ ID NO:10) is the following peptide:

1   2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 flG G G A Q V D E VV  D  I  M  R  V  N  V  D  K  V 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 L  E  R  D  Q  K  L  S  E  L  D  D  R  A  D  A  L  Q 3940 41 42 43 44 45 46 A  G  A  S  G  G  G  C

Where flG is N-fluoresceinyl-glycine. This peptide is a substrate forthe proteolytic activities of both types D and type F botulinumneurotoxins. With type D, cleavage occurs between residues 26 (K) and 27(L), while type F catalyzes hydrolysis between residues 25 (Q) and26(K).

Substrate (12) (SEQ ID NO:11) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Z N K L K S S D AY  K  K  A  W  G  N  N  Q  D  G 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 V  V  A  S  Q  P  A  R  V  V  D  E  R  E  Q  M  A  I 3940 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56S  G  G  F  I  R  R  V  T  N  D  A  R  E  N  E  M  D 57 58 59 60 61 6263 64 65 66 67 68 69 70 71 72 73 74E  N  L  E  Q  V  S  G  I  I  G  N  L  R  H  M  A  L 75 76 77 78 79 8081 82 83 84 85 86 87 88 89 90 91 92D  M  G  N  E  I  D  T  Q  N  R  Q  I  D  R  I  M  E 93 94 95 96 97 9899 100101 102 103 104 105 106 107K  A  D  S  N  K  T   R  I   D   E   A   N   Q   R 108 109 110 111 112113 114 115 116 A   T    K   M   L   G   S   G   C

Where Z is S-fluoresceinyl-cysteine. This peptide is a substrate for theproteolytic activities of types A and E botulinum neurotoxins. Type Acleaves between residues 106 (Q) and 107 (R), while type E catalyzeshydrolysis between residues 89 (R) and 90 (I).

Substrate (13) (SEQ ID NO:12) is the following peptide:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Z N K L K S S D AY  K  K  A  W  G  N  N  Q  D  G 21 22 23 24 25 26 27 28 29 30 31 32 3334 35 36 37 38 V  V  A  S  Q  P  A  R  V  V  D  E  R  E  Q  M  A  I 3940 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56S  G  G  F  I  R  R  V  T  N  D  A  R  E  N  E  M  D 57 58 59 60 61 6263 64 65 66 67 68 69 70 71 72 73 74E  N  L  E  Q  V  S  G  I  I  G  N  L  R  H  M  A  L 75 76 77 78 79 8081 82 83 84 85 86 87 88 89 90 91 92D  M  G  N  E  I  D  T  Q  N  R  Q  I  D  R  I  M  E 93 94 95 96 97 9899 100101 102 103 104 105 106 107K  A  D  S  N  K  T   R  I   D   E   A   N   Q   A 108 109 110 111 112113 114 115 116 A   T   K    M   L   G   S   G   C

Where Z is S-fluoresceinyl-cysteine. This peptide is a substrate for theproteolytic activity of type E botulinum neurotoxin. Type E catalyzeshydrolysis between residues 89 (R) and 90 (I). Replacement ofarginine-107 (see sequence in substrate (12)) with alanine preventscleavage of this substrate by type A botulinum neurotoxin.

Substrate (14):

Any peptide or protein that can serve as a substrate for the proteolyticactivity of any clostridial neurotoxin, said protein or peptide havingbeen modified so that it can be attached on one side of the proteolyticcleavage site to a solid or insoluble material. The attachment point canbe on either side (i.e. C-terminal or N-terminal) of the cleavage site.Examples of attachment methods include (but are not limited to): (a),reaction of sulfydryl groups in the substrate peptide molecules withmaleimide groups pre-attached to the solid material (or, vice versa);(b), binding of biotin groups in the substrate molecules to avidin orstreptavidin groups on the solid material (or, vice versa). Examples ofsolid materials for use in this context include, but are not limited to:(a) multiwell plastic plates; (b), plastic pins or “dipsticks”; (c),agarose beads, silica beads, plastic beads, or other types of sphericalor fibrous chromatographic media; (d), nitrocellulose or other types ofsheets or membranes.

On the other side of the cleavage site, opposite from the point ofattachment, the substrate contains a moiety that produces a measurablesignal, such as, but not limited to, a fluorescent group or aradioactive isotope. When the proteolytic activity of a clostridialneurotoxin cleaves such a substrate, the product containing the signalis released into solution. Subsequently, the amount of signal in thesoluble fraction is measured. In addition, if necessary, the amount ofresidual bound signal can also be measured following solublization ofthe latter with a protease such as trypsin.

Immobilized substrate assays have been developed for many types ofenzymes, including other proteases. However, the unusually extensivesubstrate recognition requirements of the clostridial neurotoxins andthe relatively large size of the toxin catalytic subunit (i.e. the lightchain, Mr ˜50,000) argue against the attachment of neurotoxin-specificsubstrates to solid supports. Such an arrangement would be expected toresult in considerable steric hindrance, preventing free access of thetoxin to the substrate on all sides. Furthermore, the stringentrequirements of clostridial neurotoxins with respect to substrate aminoacid sequence indicates that introduction of bulky fluorescent groups orother signal moieties into potential substrates would eliminatefunctionality. Therefore, application of knowledge gained from earlierimmobilized assays for other enzymes to the development of similarassays for clostridial neurotoxin protease activities is not encouragedand is not straightforward.

Abbreviations for the amino acids are:

-   -   A Alanine    -   D Aspartic acid    -   E Glutamic acid    -   I Isoleucine    -   K Lysine    -   L Leucine    -   M Methionine    -   N Asparagine    -   Q Glutamine    -   R Arginine    -   S Serine    -   T Threonine    -   X N-epsilon-(2,4 dinitrophenyl) lysine    -   Z S-(fluoresceinyl)-cysteine    -   dcC S-(7-dimethylamino-4-methyl-coumarin-3-carboxamidomethyl)        cysteine    -   mcp 2-amino-3-(7-methoxycoumarin-4-yl)-propionic acid    -   flG N-fluoresceinyl-glycine

Peptides can be made with commercially available automated synthesizers,using reagents and protocols obtained from the manufacturers. Aminoacids can be obtained in chemically-modified (“protected”) forms,designed so that they will react with the free amino group of thepreceding residue in the peptide chain, but not with themselves. Uponcompletion of synthesis, the peptide is cleaved from the resin,protecting groups are removed, and the product is purified. Thesepreparation protocols and others are well within the skill of a personin the art.

In another embodiment, the present invention provides a method forscreening compounds which alter BoNT activity, such as inhibitors ofBoNT activity or stimulators of BoNT activity. Solutions of BoNT orrecombinant botulinum toxin are incubated with each test compound atambient temperature, transferred to solid supports onto which isimmobilized a peptide substrate for the BoNT enzyme being tested asdescribed above, and processed as described above. A toxin incubatedwith a test compound which exhibits a reduction in the ability of thetoxin to cleave the peptide substrate relative to unincubated toxinindicates a inhibitory compound. Alternatively, a toxin incubated with atest compound which exhibits an increase in the ability of the toxin tocleave the peptide substrate relative to unincubated toxin indicated astimulatory compound.

In another embodiment, the present invention relates to a kit to searchfor compounds which inhibit or otherwise alter the protease activitiesof clostridial neurotoxins. Because the biological effects ofclostridial neurotoxins are consequences of their protease activities,it follows that compounds which affect these activities might proveuseful as anti-toxin drugs or as tools for further toxin research.Examples of compounds that could be tested include combinatorial sets ofchemicals, phage display libraries, or arrays of plant extracts. The kitwill contain in close confinement, in a box for example:

optionally, one or more underivatized multiwell plates (“preincubationplates”);

an equal number of derivatized multiwell substrate plates, which containsubstrates for clostridial neurotoxin protease activities bound to thewalls of the wells. In most cases, a well will contain substrate foronly one clostridial neurotoxin serotype, but combinations of substratesmay be used;

optionally, an equal number of opaque-wall multiwell plates, suitablefor use in a multiwell fluorimeter;

one or more of the clostridial neurotoxins or recombinant light chainsthereof;

optionally dry buffer components.

Solutions of neurotoxin or light chain in buffer are mixed with testcompounds in the same buffer in preincubation plate wells and incubatedat ambient temperature for approximately 15–30 min. This step allows thecompounds to exert effects, if any, on the toxins before exposure to thesubstrates. As controls, wells containing toxin or light chain withouttest compounds are included. Following preincubation, the solutions aretransferred to wells in the derivatized plates containing immobilizedfluorescent substrate specific for the clostridial neurotoxin beingtested. For example, if type A botulinum neurotoxin is tested, thenwells could contain the peptide described in substrate (9). Substrateplates are incubated for 1–3 hours at 30°–37° C. During this time, thetoxin or light chain protease activity will cleave the immobilizedsubstrate to a certain extent, thereby solublizing the proteolysisproduct containing the fluorescent group. Solutions are then transferredto the corresponding wells of opaque-wall plates. Fluorescence is thenquantitated in a multiwell fluorimeter. Wells containing compounds thatstimulated or inhibited toxin protease activity will have more or lessfluorescence, respectively, compared to control wells containing toxinonly.

In another embodiment, the present invention relates to a kit fordetermining the concentrations of clostridial neurotoxins in samples;for example, it may be used to monitor the various stages of botulinumtoxin production, intended for human clinical applications. Use of thiskit requires knowledge of which botulinum serotype is present, and theabsence of interfering protease(s). If these conditions are not met, useof the third kit described below is indicated. For illustrativepurposes, a kit is described for determining concentrations of type Abotulinum neurotoxin. The kit will contain in close confinement, in abox for example:

-   -   FRET substrate for type A botulinum neurotoxin, described in        substrate (2), dry;    -   optionally, dry buffer components;    -   optionally, tween-20 detergent;    -   type A botulinum neurotoxin standard.

A solution of 30 micromolar substrate is prepared in water, buffered atpH 7.3 and containing 0.05% v/v tween-20. Solutions of various knownconcentrations of type A botulinum neurotoxin are prepared in the samebuffer. Toxin is mixed with substrate, the increase in fluorescence ismeasured for a period of time, and the initial rate of fluorescenceincrease is determined from the early (essentially linear) part of thecurve. This is repeated for each known concentration of toxin,establishing a correlation between toxin concentration and rate.Cleavage rates are then determined for samples containing unknownconcentrations of type A neurotoxin. By comparison with the standards,the unknown concentrations may be calculated.

In another embodiment, the present invention relates to a kit fordetecting the presence of clostridial neurotoxins in samples. Inaddition to detection, the kit will also eliminate interfering proteasesthat might be present, identify the serotypes of the neurotoxins, andpermit calculations of neurotoxin concentrations. Kits may be used toscreen just a few samples, or large numbers of samples at once. The kitwill contain in close confinement, in a box for example:

a multiwell plate, containing biotinylated antibodies against allserotypes of the clostridial neurotoxins, bound to avidin- orstreptavidin-coated wells. The antibodies are specific for the heavychains of the toxins. Generally, a particular well would containantibodies against only one of the clostridial neurotoxins, but allwould be represented on the plate. However, in cases where volume oftest sample is limited, wells could contain more than one type ofantibody. The plate is preferably suitable for use in a multiwellfluorimeter;

-   optionally, dry buffer components for wash buffer;-   optionally, dry activation buffer components, containing buffer,    dithiothreitol, and zinc chloride;-   optionally, Tween-20 detergent;-   Clostridial neurotoxin standards.-   Type (I) FRET substrates and/or type (II) substrates, the latter    preferably immobilized, for example, by attachment to derivatized    plastic pins which are commercially available. The pins can be used    individually, or may be attached to plate lids in an array that    corresponds to that of the plate wells, such that, when a lid is    applied to a plate, one pin enters each well.

Solutions of samples suspected of containing clostridial neurotoxins areplaced in plate wells. Wells containing antibodies against eachclostridial neurotoxin should receive sample, but if sample volume islimited, wells containing multiple anti-neurotoxin antibodies would beused. Plates are incubated for approximately one hour at 30°–37° C.During this time, clostridial neurotoxins, if present, will bind to theanti-toxin antibodies in the wells. Wells are then washed with washbuffer (typically, 50 mM tris, 0.1% v/v tween-20, pH 7.5, but others maybe used) to remove unbound components including, if present, proteasesother than the clostridial neurotoxins. Activation solution (20 mMbuffer, 10 mM dithiothreitol, 0.50 mM zinc chloride. pH 7.3) is addedand incubated at 30°–37° C. for 20–30 minutes to activate clostridialneurotoxin protease activities. Solutions containing FRET substrates areadded, corresponding to the type of neurotoxin that would be captured bythe antibody in a particular well. After one hour incubation at 30°–37°C., the amounts of fluorescence in the wells are determined with afluorimeter. By comparing fluorescence readings in test sample wells tothose obtained from control wells (buffers and substrates only) and toreadings from wells containing neurotoxin standards, the presence of aclostridial neurotoxin in a sample may be detected, and itsconcentration determined. By noting the specificity of the antibody in apositive well, the serotype of the clostridial neurotoxin is revealed.

Alternatively, plastic pins derivatized with the appropriate type (II)immobilized substrates are placed in the wells, instead of type (I) FRETsubstrates. (Circumstances favoring the use of one substrate type overthe other are discussed in a preceding section). After incubation, thepins are removed. If the pins were arrayed in a plate lid, correspondingto the well array, this is accomplished simply by removing the lid. Theamount of fluorescence in the wells is then determined in a multiwellfluorimeter. Calculations of results are done as discussed above.

In situations where wells containing antibodies against more than oneserotype must be used, differentiation among neurotoxin serotypes isenabled by using substrates containing different fluorophores. Forexample, a well might contain antibodies against botulinum serotypes A,B, E, and F. After incubation of sample, washing, and activation, amulti-substrate plastic pin with a combination of four immobilized type(II) substrates is placed in the well. In this example, the pin isderivatized with: (1) substrate for botulinum type A described insubstrate (9); (2) substrate for botulinum type B described in substrate(10), modified to replace N-fluoresceinyl-glycine withN-rhodaminyl-glycine as the N-terminal moiety; (3) substrate forbotulinum type E described in substrate (13), modified to replaceS-fluoresceinyl-cysteine with N-(7-methoxy-coumarin-4-acetyl)-glycine asthe N-terminal moiety; (4) substrate for botulinum type F described insubstrate (11), modified to replace N-fluoresceinyl-glycine withS-(4-acetamido-stilbene-2-2′-disulfonicacid-4′-carboxamidomethyl)-cysteine. Because the four fluorophores havedistinct and well-separated emission spectra, the presence of one ormore botulinum serotype(s) is/are revealed by determining thewavelength(s) of any fluorescence remaining in the well after theremoval of the plastic pin.

Immobilization of clostridial neurotoxin serotype-specific antibodiesmay be accomplished in other ways. For example, instead of substrates,the antibodies are bound to plastic pins or dipsticks, for use eitherindividually or in arrays corresponding to multiwell plates. The pinsare immersed in test samples. If clostridial neurotoxins are present,they will be captured by the antibodies. After rinsing, the pins areimmersed in solutions containing activation buffer (see above), followedby addition of type (I) FRET substrates. An increase in fluorescence(compared to appropriate controls) indicates the presence of neurotoxin.The serotype is revealed by the type of antibody on the pin. Theconcentration may be calculated from a standard curve constructed withknown concentrations of neurotoxin.

Alternatively, plastic pins derivatized with the appropriate type (II)immobilized substrates are placed in the wells, instead of type (I) FRETsubstrates. (Circumstances favoring the use of one substrate type overthe other are discussed in a preceding section). After incubation(v.s.), the pins are removed. If the pins were arrayed in a plate lid,corresponding to the well array, this is accomplished simply by removingthe lid. The amount of fluorescence in the wells is then determined in amultiwell fluorimeter. Calculations of results are done and conclusionsdrawn as discussed above.

In another modification, neurotoxin-specific antibodies are bound to thesurface of a sheet or membrane in roughly circular spots, correspondingto a multiwell plate array. The sheet or membrane is immersed in thetest sample, followed by rinsing. The sheet is then clamped between theupper and lower halves of a multiwell plate, such that the sheet formsthe bottom of each well. Alternatively, instead of immersing the sheetin the sample, test samples may be added to the wells, then washed out,or removed by drawing through the sheet by application of a vacuum tothe lower plate half. Activation buffer and then type (I) FRETsubstrates are added to the wells. Results are calculated andconclusions drawn as noted above. Multiwell plates consisting ofseparable upper and lower halves or chambers, with and without thecapability of applying vacuum to the lower chamber, are commerciallyavailable and not specifically claimed in this patent application.

The following examples are illustrative of the practice of the inventionbut should not be read as limiting the scope thereof. It is understoodthat various modifications could be suggested within the spirit andpurview of this application and the scope of the claims.

The following materials and methods were used in the examples below.

Materials and Methods

Enzyme Preparations

Botulinum toxins were obtained from Food Research Institute, Madison,Wis. All preparations appeared to be more than 90% pure, as judged bySDS-PAGE under reducing conditions. Botulinum toxins were used only byimmune personnel under Biosafety Level-2 controls, in accordance withthe recommendations of the U.S. Centers for Disease Control andPrevention [Biosafety in Microbiological and Biomedical Laboratories(1999) U.S. Government Printing Office, Washington, D.C.].Maleimide-activated 96-well plates were purchased from Pierce ChemicalCo., Rockford, Ill.

Recombinant botulinum type A light chain was expressed in competent E.coli BL21 cells (Stratagene, La Jolla, Calif.) using a synthetic gene[Ahmed and Smith (2000) J. Prot. Chem. 19: 475–487] and a pET vectorbased upon the T7 RNA polymerase expression system Studier et al. (1990)Methods Enzymol. 185: 60–89]. Soluble recombinant type A light chain waspurified to >90% purity by ion exchange chromatography at roomtemperature [Li and Singh (1999) Prot Expr. Purif. 17: 339–344]. 12:31PM

Peptide Synthesis

The peptide synthesizer was a model 431A from Perkin Elmer-AppliedBiosystems, Foster City, Calif. We used protocols, reagents, andchemicals obtained from the manufacturer. Rink resin was employed toyield a carboxamide at the C-terminal residues of all peptides.N(alpha)-FMOC-N(epsilon)-2,4-dnitrophenyl-lysine andN-FMOC-2-amino-3(7-methoxycoumarin-4-yl)-propionic acid were purchasedfrom Bachem Bioscience, King of Prussia, Pa.

Substrates with S-fluoresceinyl cysteine were prepared by reacting thecysteine sulfhydryl group in the substrate with iodoacetamidofluorescein(Pierce Chemical Co., Rockford Ill.). Similarly, substrates containingS-(7-dimethylamino-4-methyl-coumarin-3-carboxamidomethyl)-cysteine wereprepared by reaction of the cysteine sulfhydryl group withN-(7-dimethylamino-4-methylcoumarin-3-yl)iodoacetamide (MolecularProbes, Eugene Oreg.).

Fluorescein was coupled to N-terminal glycine by deblocking thealpha-amino group, followed by reacting the resin-bound protectedpeptide with 5-carboxyfluorescein-N-hydroxysuccinimide ester (PierceChemical Co., Rockford, Ill.). Alternatively, 5-carboxy-fluorescein(Aldrich Chemical Co., Milwaukee, Wis.) was loaded into synthesizercartridges and placed on the instrument as the last residue. Twocouplings were required for complete reaction. After deprotection andcleavage from the resins, crude peptides were purified by reverse-phaseHPLC.

Preparation of Immobilized-Substrate Assay Plates

For covalent coupling to maleimide-activated multiwell plates,fluorescent peptides were dissolved at concentrations of 15 to 20 μM in50 mM Tris, 5 mM EDTA, 0.1% tween, pH 7.9. All wells received 100 μL ofpeptide solution, followed by incubation at ambient temperature for 4–5h. Wells were emptied, then incubated in succession (200 μL per well, 1h) with 40 mM 2-mercaptoethanol in Tris-EDTA-tween, then 10 mg/ml BSA inTris-tween (no EDTA). Finally, wells were washed three times with 200 μLof Tris-tween. Assay plates were stored dry at −10° to −20° C.

Coupling of Substrates to Derivatized Plastic Pins

Derivatized pins (also called “gears”) can be obtained from Mimotopes,Raleigh N.C., with N-FMOC-beta-alanine bound through a spacer moiety tothe polymer backbone. After removal of the FMOC with piperidine, thefree amino group of the beta-alanine is iodoacetylated by reaction withiodoacetic anhydride. Substrate peptide is then covalently bound byreaction of the sulfhydryl group in the substrate cysteine residue withN-iodoacetyl-beta-alanine on the pins.

Assays of Botulinum Toxin Proteolytic Activity

Botulinum toxins (10–20 μg/ml) were preactivated by incubation at 37° C.for 30 min in 20 mM hepes, pH 7.3, 10 mM DTT, 0.50 mM ZnCl₂, and 0.05%tween. Assays were conducted in 20 mM hepes, 5 mM DTT, 0.25 mM ZnCl₂,and 0.05% tween, with various concentrations of toxins.

When recombinant type A light chain was employed in the assays, thelyophilized enzyme was reconstituted to 35–50 ug/ml with 40 mMhepes/0.05% tween, pH 7.3, followed by dilution to the desiredconcentraion in the same buffer. Unlike whole toxin, preactivation oflight chain is not necessary. Botulinum toxin or recombinant type Alight chain were incubated in the substrate-coated plates for varyingtimes at 35° C. in the dark without agitation. Appropriate control wellscontaining buffer only were included on each plate. Because themaleimide-activated plates have clear walls, they are not optimized fordirect fluorescence measurements. Therefore, after incubation, aliquotswere transferred to corresponding wells of opaque-wall plates, andfluorescence was measured with a Wallac 1420 multiwell fluorimeter(PerkinElmer Wallac, Gaithersburg Md., USA). Excitation and emissionwavelengths were 485 and 535 nm, respectively, with readings of 1 secondper well. All fluorescence values were expressed in arbitrary units asthe mean of triplicate determinations. Error bars are standarddeviations.

Assays employing FRET substrates were done in cuvettes in atemperature-controlled fluorimeter (Photon Technology International,Newtown, Pa.). Toxin or light chain was mixed with appropriate FRETsubstrate, placed in the instrument, and fluorescence was measured forvarious times.

EXAMPLE 1

Operation of the Invention Using a Type (I) Substrate:

In this case, the substrate was the peptide described in substrate (1)above, and the enzyme was a recombinant preparation of type A botulinumtoxin catalytic domain (also known as “type A light chain”). A solutionof 30 micromolar peptide was prepared in water, buffered at pH 7.3, andcontaining 0.05% v/v tween-20. Before addition of enzyme, fluorescencewas measured to obtain the background or “zero-time” fluorescence.Enzyme was then added to a concentration of two micrograms per ml, andthe resulting increase in fluorescence due to proteolysis of the peptidewas measured with time. Assay temperature was 21° C. In the absence ofenzyme, fluorescence changed very little with time, less than ±5%.Results are shown in FIG. 1. The initial rate of hydrolysis can becalculated from the slope of the line in the early (essentially linear)part of the curve. Using known concentrations of toxin or light chain, acorrelation between rate and concentration can be established, allowingcalculation of toxin or light chain concentrations in unknown samples.

EXAMPLE 2

Operation of the Invention Using Type (II) Substrates:

In this example, the substrates were the peptides described in substrate(9) for botulinum type A, substrate (10) for type B, and substrate (11)for types D and F. The “solid material” to which the substrates wereimmobilized were 96-well microtiter plates that were chemically modifiedto contain maleimide groups (see “Materials and methods” section). FIG.2 depicts fluorescence solubilized by different concentrations ofbotulinum toxins, serotypes A, B, D, and F (3 hours, 35° C.). For eachserotype, maximum fluorescence was defined as that solubilized by 1microgram per ml toxin under these conditions. The Y-axis extends above100% to accomodate 12:31 PM the error bars. Panel (a) shows BoNT A andB; panel (b) shows BoNT D and F.

It is understood that these descriptions, examples and embodiments arefor illustrative purposes only, and that various modifications would besuggested within the spirit and purview of this application and thescope of the appended claims.

1. A botulinum neurotoxin serotype B or tetanus toxin substratecontaining a fluorescent signal moiety on one side of the cleavage sitethat produces a fluorescent signal and a moiety that quenches themagnitude of said signal on the other side of the cleavage site suchthat when the substrate is cleaved, an increase in fluorescent signal isproduced, wherein said substrate is a peptide identified as SEQ ID NO:3or SEQ ID NO:4.
 2. A kit for determining the concentration of botulinumneurotoxin serotype B or tetanus toxin in a sample, the kit containingin close confinement: (i) one or both peptide substrates according toclaim 1 cleavable by said botulinum neurotoxin or said tetanus toxin;and (ii) said botulinum neurotoxin standard or said tetanus toxinstandard.
 3. A method for measuring the concentration of botulinumneurotoxin serotype B or tetanus neurotoxin in a sample, said methodcomprising: mixing the sample with a peptide substrate identified as SEQID NO:3 or SEQ ID NO:4, measuring an increase in fluorescent signal withtime produced from proteolytic cleavage of said substrate, anddetermining the concentration of said neurotoxin by correlating to astandard of said neurotoxin.
 4. A method for detecting the presence ofbotulinum neurotoxin serotype B or tetanus toxin proteolytic activity ina sample, said method comprising: mixing the sample with a peptidesubstrates identified as SEQ ID NO:3 or SEQ ID NO:4, and detecting anincrease in fluorescent signal produced from proteolytic cleavage ofsaid substrate.
 5. A botulinum neurotoxin serotype B or tetanus toxinsubstrate comprising a peptide which is optionally immobilized to asolid material and which contains a fluorescent moiety that produces ameasurable fluorescent signal such that when the substrate is cleaved,the fluorescent signal is released, wherein said substrate is a peptideidentified as SEQ ID NO:9.
 6. A kit for determining the concentration ofbotulinum neurotoxin serotype B or tetanus toxin in a sample, the kitcontaining in close confinement: (i) the peptide substrate according toclaim 5 cleavable by said botulinum neurotoxin or said tetanus toxin;and (ii) said botulinum neurotoxin or said tetanus toxin standard.
 7. Amethod for detecting the presence of botulinum neurotoxin serotype B ortetanus toxin proteolytic activity in a sample, said method comprising:mixing the sample with a peptide substrate identified as SEQ ID NO:9,and detecting an increase in fluorescent signal produced fromproteolytic cleavage of said substrate.
 8. A method for measuring theconcentration of botulinum neurotoxin serotype B or tetanus toxin in asample, said method comprising: mixing the sample with a peptidesubstrate identified as SEQ ID NO:9, measuring an increase influorescent signal with time produced from proteolytic cleavage of saidsubstrate, and determining the concentration of said neurotoxin bycorrelating to a standard of said neurotoxin.
 9. A method foridentifying a compound that inhibits or enhances the proteolyticactivity of botulinum neurotoxin serotype B or tetanus neurotoxin, saidmethod comprising: preincubating the neurotoxin with a test compound tomake a neurotoxin-compound solution, exposing said solution to a peptidesubstrate identified as SEQ ID NO:9, measuring fluorescent signalresulting from proteolytic cleavage of said substrate by saidneurotoxin, and comparing said fluorescent signal of theneurotoxin-compound solution with a control, wherein the control is theneurotoxin solution without the presence of the test compound, andwherein an increase in fluorescent signal indicates a compound thatenhances neurotoxin activity and a decrease in fluorescent signalindicates a compound that inhibits said neurotoxin.